Immunoassay method for lyzed whole blood

ABSTRACT

The present invention provides an immunoassay method in which blood can be measured even without pretreatment by means of a centrifuge etc. In the present invention, antibodies or antigens in a sample are subjected to agglutination reaction with insoluble carriers onto which antigens or antibodies specifically reacting with the antibodies or antigens in the sample have been immobilized and the resulting agglutination mixture is determined for the change in its absorbance or in its scattered light by irradiation with light, wherein said sample is whole blood and the whole blood is forcibly lyzed.

This is a divisional application of U.S. Ser. No. 08/914,039, filed onJul. 28, 1997 now U.S. Pat. No. 6,030,845.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relate immunoassay method and in particular to animmunoassay method for use with lysed whole blood in which antibodies orantigens in a sample are subjected to agglutination reaction withinsoluble carriers onto which antigens or antibodies specificallyreacting with the antibodies or antigens in the sample have beenimmobilized. The resulting agglutination mixture is irradiated with nearinfrared or infrared rays to determine its change in absorbance or itschange in scattered light.

2. Description of Related Art

Japanese Patent Publication No. 11575/1983 discloses a prior art methodwhich comprises antigen-antibody reaction between antigen- orantibody-immobilized insoluble carriers and antibodies or antigens in ahumor sample, then irradiating the reaction mixture with light with awavelength of 600 to 2400 nm and measuring the increase in itsabsorbance. By virtue of its usefulness, this method has become themainstream of immunoassay method at present as a so-called lateximmunoturbidimetry.

However, the measurement sample used in said measurement method iswater, serum, urine, saline etc. In addition, matters that requireattention in general blood taking for clinical examination are thathemolysis should be avoided to a maximum degree and blood should beseparated into serum and plasma as rapidly as possible. The reasons forthis include the effect of hemolysis on optical measurement, theincoming and outgoing of substances such as Na, K, C1 through bloodmembrane, the effect of movement by blood corpuscles metabolism (i.e.,transfer of lactic acid and pyruvic acid to serum by glycolysis) and theeffect of the difference in concentration of the object component inblood corpuscles and in serum.

For the above reasons, blood obtained from a subject should be a sampleseparated into serum or plasma by centrifugation. Therefore, suchpretreatment by centrifugation may not be carried out in small orprivate laboratories or urgent laboratories other than centrallaboratories in large or middle hospitals where a large amount of bloodcan be dealt with, and therefore the above method is not necessarilyuniversal.

Under the circumstances of such general whole blood handling, in thefield of clinical examination there is no accurate and quantitativemeasurement method in which whole blood can be directly used as ameasurement sample without separating it into serum and plasma. Further,the measurement of blood using optical means without hemolysis isinappropriate because of high turbidity caused by erythrocyte.

OBJECTS AND SUMMARY OF THE INVENTION

In view of the foregoing, the present invention was made, and a firstobject is to provide an immunoassay method which can be effected easilyin a short time even without preteating blood by, e.g., a centrifuge,and a second object is to provide an immunoassay method using wholeblood directly as a sample in which blood corpuscles are subjectedintentionally to forcible hemolysis in a manner not to affectimmunoreaction so that accurate data can be obtained in combination withvarious quantitative measurement reagents.

As a result of their research, the present inventors unexpectedly foundthat antigens or antibodies in whole blood can be determined bysubjecting whole blood intentionally to forcible hemolysis in a mannernot to affect agglutination reaction, as opposed to the fixed concept ofgeneral blood taking for clinical examination, that is, hemolysis shouldbe avoided to a maximum degree and blood should be separated into serumand plasma as rapidly as possible.

To achieve the first object, the present invention comprises animmunoassay method in which antibodies or antigens in a sample aresubjected to agglutination reaction with insoluble carriers onto whichantigens or antibodies specifically reacting with the antibodies orantigens in the sample have been immobilized and the resultingagglutination mixture is determined for the change in its absorbance orin its scattered light by irradiation with light, wherein said sample iswhole blood and the whole blood is forcibly lyzed.

In this case, the means of forcible hemolysis can include:

-   -   (1) mixing whole blood with a low osmotic solution;    -   (2) mixing blood with a solution of saponins for hemolysis;    -   (3) freezing and thawing whole blood; and    -   (4) ultrasonicating whole blood.

Alternatively, saponins for hemolysis may be incorporated into aninsoluble particle suspension reagent onto which antibody or antigenspecifically reacting with antigen or antibody has been immobilized.

To achieve the second object, the immunoassay method according comprisesthe step of subjecting antibodies or antigens in whole blood as a sampleto agglutination reaction with an insoluble particle suspension reagentonto which antigens or antibodies specifically reacting with theantibodies or antigens in the whole blood have been immobilized, thestep of determining the resulting agglutination mixture for the changein its absorbance or in its scattered light by irradiation with light,and the step of calculating the hematocrit % of as follows by thesample:A′=A×100/(100−hematocrit %)where A is the absorbance or its change or the strength of lightscattering or its change actually determined, and A′ is the correctedabsorbance or its change or the strength of light scattering or itschange assuming that the plasma component in the sample is 100%.

According to the first object, the following effects are achieved:

-   -   (1) By using a whole blood sample directly in the measurement        procedure without subjecting it to pretreatment such as        centrifugation etc., measurement time can be shortened,        measurement costs can be reduced, and a measurement operation        can be simplified. Because centrifugation is not required, the        costs for a centrifuge or centrifuge tube, the operation for        transferring a sample to a centrifuge tube, the time for        centrifugation can be eliminated, and the opportunity for an        operator to come into contact with blood can be decreased        whereby the danger of infection can be significantly reduced.    -   (2) By subjecting blood corpuscles in whole blood to forcible        hemolysis in a manner not to cause any effect on        antigen-antibody reaction, the method can be combined with a        measurement kit using general latex immunoturbidimetry whereby        accurate measurement data can be obtain while a wide variety of        applications is made feasible.    -   (3) By incorporating a hemolysis reagent into a latex reagent,        the constitution of the measurement apparatus can be simplified        and the measurement time can be reduced.

According to the present invention, accurate data can be obtained byhematocrit correction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing an example of the cell used in the method ofthe present invention.

FIG. 2 is a schematic drawing showing the constitution of thespectrophotometer used in the method of the present invention

FIG. 3 is a drawing showing an example of the freezing cell holder usedin the method of the present invention.

FIG. 4 is a drawing showing an example of the ultrasonication nozzleused in the method of the present invention.

FIG. 5 is an absorption spectrum at 300 to 1000 nm of whole blood havingbeen subjected to hemolysis with aqueous solutions of various hemolysisreagents

FIG. 6 is a drawing showing hemolysism reaction time course at 800 nm ofwhole blood having been subjected to hemolysis with aqueous solutions ofvarious hemolysis reagents.

FIG. 7 is a calibration curve of change in absorbance per minute as afunction of CRP concentration obtained when CRP measurement is carriedout.

FIG. 8 is a graph showing a correlation between a whole blood sample anda plasma sample when no hematocrit correction is carried out.

FIG. 9 is a graph showing a correlation between a whole blood sample anda plasma sample when hematocrit correction is carried out

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail by referenceto Examples.

The reagents examined in the Example are shown in Table 1 below. Thesymbols “a” to “g” in Table, 1 are identical with those in FIGS. 5 to 7.

TABLE 1 Hemolysis Reagent Absor- ΔAbsor- Aqueous Content bance bance/Solution Hemolysis Method (W/V %)* (800 nm) min a Pure Water 0.204 0.003(Deionized Water b Saponin Aqueous Solution 0.5 0.147 0.000 c TRITONX-100 0.5 0.146 0.000 (Non-Ionic Surface Active Agent) d TWEEN-20 0.50.298 0.055 (Non-Ionic Surface Active Agent) e BRIJ 35 0.5 2.312(Non-Ionic Surface Active Agent) f Sodium Lauryl Sulfate 0.5 0.176 0.000(Anionic Surface Active Agent) g Benzalkonium Chloride 0.5 0.139 0.000(Cationic Surface Active Agent) h Freezing Hemolysis 0.163 0.000 iUltrasonication Nozzle 0.196 0.001 Hemolysis j Physiological Saline3.000 *W/V % = weight/volume %

[Example 1] Hemolysis by Hemolysis Reagents

0.04 ml of human whole blood collected in a usual manner using anEDTA-2K anticoagulant was placed in quartz cell 5 with a cell length of5 mm as shown in FIG. 1. 2.0 ml of each of Hemolysis regent aqueoussolutions “a” to “g” shown in Table 1 was added to it and monitored forits absorption spectrum at 300 to 1000 nm (see FIG. 5) inspectrophotometer 1 (e.g., U-3410, produced by Hitachi, Ltd., Japan),for its Hemolysis reaction time course at 800 nm (see FIG. 6), for itsabsorbance 5 minutes after the reaction was initiated and for the changein its absorbance at 800 nm for 1 minute between 4 and 5 minutes afterthe reaction was initiated (see Table 1) to examine the ability of eachhemolysis reagent to lyse blood.

In FIG. 2, 2 is a halogen lamp as a light source emitting irradiationlight L, e.g., near infrared or infrared rays; 3 is a condensing lens; 4is a diffraction grating; 6 is an amplifier; and 7 is an arithmetic andrecording device such as computed and 8 is a display. “S” is whole bloodhaving been subjected to hemolysis treatment as a sample accommodated incell 5.

As shown in FIG. 5 non-lysed blood treated with the reagent “j”(physiological saline) showed an absorbance of 2.5 or more due to itsturbidity at the shown wavelengths, resulting in affecting the opticaldetection of latex agglutination reaction. On the other hand, it wasfound that as shown in FIG. 5, such turbidity as described abovedisappeared by use of the reagent “a” (pure water) and the reagent “b”(saponin aqueous solution) so that the degree of agglutination of latexcan be determined. As can be seen from Table 1 and FIG. 6, the reagent“a” (pure water), the reagent “b” (saponin), the reagent “o” (TritonX-100), the reagent “f” (sodium lauryl sulfate), and reagent “g”(benzalkonium chloride) have the ability to lyse whole blood in a shorttime.

Example 2 Hemolysis by Freezing

FIG. 3 shows one embodiment of freezing cell holder 9 for use in lyzingwhole blood, which can receive and maintain cell 5 in it and includes aPeltier element 12 (a product of, e.g., Merukoa Co., Ltd.) attached tocell block 11 made of aluminum provided with photometric window 10. 13is a power source for applying a direct current as necessary to Peltierelement 12, and L is a near infrare or infrared ray from the powersource 2. 0.04 ml of human whole blood collected in a usual manner usingan EDTA-2K anticoagulant was accommodated in cell 5 placed in freezingcell holder 9 as shown in FIG. 3, and the human whole blood wascompletely frozen by applying an electric current to the Peltier element12 for 10 minutes in a predetermined direction. Thereafter, the frozenhuman whole blood was thawed by applying an electric current in thereverse direction to the Peltier element 12, then diluted with 2.0 mlphysiological saline, and examined for its absorbance 5 minutes afterthe reaction was initiated and for the change in its absorbance at 800nm for 1 minute between 4 and 5 minutes after the reaction was initiated(see Table 1) to determine the ability of the hemolysis reagent to lyseblood. As can be seen in “h” in Table 1, human whole blood can be lysedby freezing and thawing.

[Example 3] Hemolysis by Ultrasonication

FIG. 4 shows one embodiment of ultrasonic nozzle 14 for use in lysingwhole blood, which includes ultrasonic radiator 16 attached to stainlesssteel nozzle 15, and 17 is a radiator circuit, and 18 is a suctionsyringe.

0.04 ml of human whole blood obtained in a usual manner using an EDTA-2Kanticoagulant was suctioned into nozzle 15, and the ultrasonic radiator16 was run for 5 minutes to lyse human whole blood B in nozzle 15.Thereafter, the lysed human whole blood B was accommodated in nozzle 15,then diluted with 2.0 ml physiological saline, and examined inspectrophotometer 1 for its absorption at 800 nm 5 minutes after thereaction was initiated and for the change in its absorbance for 1 minutebetween 4 to 5 minutes after the reaction was initiated (see Table 1) todetermine the ability of the hemolysis reagent to lyse blood. As can beseen from symbol “i” in Table 1, human whole blood can be lysed byultrasonicating it.

[Example 4] C-Reactive Protein (CRP) Measurement Method 1

1) Preparation of Anti-CRP Antibody Solution

An about 10 mg/ml anti-human CRP (C-reactive protein) rabbit antibodysolution (pH 7.5, 100 mmol/l Tris-HCl buffer, 0.1% sodium amide) wasadded to 10 ml polystyrene latex with an average particle diameter of0.2 pun (produced by, e.g., Japan Synthetic Rubber Co., Ltd., Japan; 10%solid content), and the mixture was allowed to stand at 30° C. one wholeday and night and then centrifuged at 3600 rpm to give precipitates. 0.2W/V % bovine serum albumin, pH 8.5, 100 mmol/l Tris-HCI buffer was addedto the precipitates to prepare an anti-CRP antibody sensitized latexsolution.

2) CRP Measurement Method

0.04 ml of human whole blood collected in a usual manner using anEDTA-2K anticoagulant was placed in cell 1, and 0.5 ml of each ofhemolysis regent aqueous solutions “a” to “g” shown in Table 1 was addedto it, and the mixture was incubated at 37° C. for 3 minutes, and 1.5 mlof the anti-human CRP antibody sensitized latex suspension prepared initem 1) above was added to it, and the change in its absorbance at 800nm for 1 minute between 4 and 5 minutes after the reaction was initiatedwas determined.

Separately, a calibration curve of the above sample was prepared using acommercial latex immunoturbidimetry CRP measurement kit intended for usein examining plasma as its sample. FIG. 5 shows a calibration curveprepared using the results obtained in the above CRP measurement, and acalibration curve excellent in sensitivity as shown in symbols “a” and“b” in the figure was obtained using whole blood lyzed forcibly withpure water “saponin aqueous solution “b” etc. However, the resultsindicated that surface active agents “c” to “g” inhibit agglutinationreaction and are thus not suitable for immunoreaction, as shown in thesymbols “c” to “g” in the figure.

[Example 5] CRP Measurement Method Using Blood Sample Lyzed by Freezingor Ultasonication

The operation of dilution with physiological saline after hemolysis inExample 2 or 3 was replaced by addition of 2.0 ml of the anti-human CRPantibody sensitized latex suspension prepared in Example 4, and a changein absorbance at 800 nm for 1 minute between 4 to 5 minutes after thereaction was initiated was determined in spectrophotometer 1.

Separately, a calibration curve of the above sample was prepared using acommercial latex immunoturbidimetry CRP measurement kit intended for usein examining serum or plasma as its sample. As shown in symbols “h” and“i” in FIG. 7, a calibration curve excellent in sensitivity wasobtained.

Example 6 CRP Measurement Method 2

The same measurement method as in Example 4 was used except that acommercial latex immunoturbidimetry CRP measurement kit was used inplace of the anti-human CRP antibody sensitized latex suspension used inExample 4 and 0.5 w/v S saponin aqueous solution was used as a hemolysisreagent in order to determine a change in absorbance at 800 nm for 1minute between 4 to 5 minutes after the reaction was initiated. Acomparison between a calibration curve (n=40) using measurement valuesof whole blood as a sample determined according to the present inventionand a calibration curve using measurement values of serum as a sampledetermined according to a general method indicated good correlation asshown in FIG. 8.

Example 7 Hematocrit Correction

To correct the measurement values obtained according to the presentinvention in Example 6, the whole blood was simultaneously determinedfor its hematocrit value by means of an erythrocyte counter (e.g.,LC-240A manufactured by Horiba Seisakusho K. K., Japan) using thefollowing formula (1):A′=A×100/(100−hematocrit %)  (1)where A is the actually determined change in absorbance, and A′ is itscorrected change assuming that the plasma component in the sample is100%. A comparison (n=40) between the measurement values thus correctedand the measurement values determined in a general method using serum asa sample indicated further improved correlation as compared with that ofExample 6, as shown in FIG. 9.

In the Examples above, the change in absorbance of an agglutinationmixture by light irradiation was determined; alternatively, the changein scattered light may be determined.

The present invention is practiced in the embodiments described aboveand demonstrates the following effects:

According to the present first invention, measurement time can beshortened, measurement costs can be reduced, and measurement operationcan be simplified because a whole blood sample can be used directlywithout subjecting it to pretreatment such as centifugation, etc.Further, the opportunity for an operator to come into contact with bloodcan be decreased and the danger of infection can be significantlyreduced.

According to the present second invention, accurate data can be obtainedby conducting hematocrit correction.

1. An agglutination immunoassay method of quantifying a predeterminedantigen in a sample of whole blood, comprising the steps of: providing asample of the whole blood: adding a hemolysis reagent and a latexreagent comprising of insoluble latex carriers onto which antibodiesspecifically reacting with the predetermined antigen in the sample ofwhole blood have been immobilized, directly to the sample of the wholeblood without any pre-treatment of the whole blood; hemolysing the wholeblood sample with the hemolysis reagent to hemolyse the bloodcorpuscles; forming a reaction product wherein a predetermined antigenin the hemolysed whole blood sample specifically reacts with theantibodies immobilized onto the insoluble latex carriers; irradiatingthe reaction products in the sample with radiation which includes awavelength within a range of 700 nm to 1000 nm which is substantiallyfree from absorption by both hemoglobin and the hemolysis reagent; andmeasuring only in the wavelength range which is substantially free fromabsorption by both hemoglobin and the hemolysis reagent, an absorbanceof the incident radiation through the reaction product to determine thequantity of antigens in the sample.
 2. The immunoassay method of claim1, wherein the step of hemolysing is performed with a saponin aqueoussolution as the hemolysis reagent.
 3. The immunoassay method of claim 1wherein the wavelength approximately 800 nm.
 4. An agglutinationimmunoassay method of quantifying a predetermined antigen in a sample ofwhole blood, comprising the steps of: providing a sample of the wholeblood; adding a hemolysis reagent and a latex reagent, includinginsoluble later carriers onto which antibodies specifically reactingwith the predetermined antigen in the sample of whole blood have beenimmobilized, directly to the sample of the whole blood without anypre-treatment of the whole blood; hemolysing the whole blood sample withthe hemolysis reagent to hemolyse the blood corpuscles; reacting thehemolysed whole blood sample in an agglutination reaction to form anagglutination reaction product wherein a predetermined antigen in thehemolysed whole blood sample specifically reacts with the antibodiesimmobilized onto the insoluble latex carriers; irradiating theagglutination reaction product in the hemolysed whole blood sample withradiation which includes a wavelength within a range of 700 nm to 1000nm which is substantially free from absorption by both hemoglobin andthe hemolysis reagent; and measuring, only within the wavelength rangeof 700 nm to 1000 nm, an absorbance of the incident radiation with theagglutination reaction product to determine the quantity of antigens inthe sample.
 5. The agglutination immunoassay method of claim 4 furtherincluding the step of determining the C-reactive protein (CRP) componentin plasma in the hemolysed whole blood sample.
 6. The agglutinationimmunoassay method of claim 4 wherein the wavelength is approximately800 nm for measuring.
 7. The agglutination immunoassay method of claim 4wherein the hemolysing reagent is saponin.
 8. A particle agglutinationimmunoassay method of quantifying a predetermined antigen in a sample ofwhole blood, comprising the steps of: providing a sample of the wholeblood; adding a hemolysis reagent to the sample of whole blood;hemolysing blood corpuscles in the sample of whole blood to enable asubsequent immunoreaction; adding a latex reagent, including insolublelatex carriers onto which antibodies specifically reacting with thepredetermined antigen in the sample of whole blood have beenimmobilized, to the hemolysed whole blood; providing an agglutinationreaction with the hemolysed whole blood sample to form an agglutinationreaction product wherein a predetermined antigen in the hemolysed wholeblood sample reacts with the antibodies immobilized on the insolublecarriers to provide the agglutination reaction product; irradiating theagglutination reaction product in the hemolysed whole blood sample withradiation which includes a wavelength of approximately 800 nm which issubstantially free from absorption by both hemoglobin and the hemolysisreagent; and measuring, only with the wavelength of approximately 800nm, a change in absorbance of the incident radiation by theagglutination reaction product to determine the quantity of antigens inthe sample.
 9. The particle agglutination immunoassay method of claim 8wherein the hemolysing reagent is saponin.
 10. The particleagglutination immunoassay method of claim 8 wherein the predeterminedantigen is the C-reactive protein (CRP) composed in plasma in thehemolysed whole blood sample.